Essentially every step in the HIV life cycle interfaces intimately with the host cell machinery. The Pittsburgh Center of HIV Protein Interactions will focus on the steps and interactions that occur with the host after engagement of cell surface receptors and membrane fusion and before integration of the viral genome into that of the host, the so called early events. Several essential molecular interactions and enzymatic activities occur within this time window, necessary for productive progression of the viral life cycle. Thus, it represents a pivotal period in the infection process, during which the susceptibility of the virs to disruptive interventions is likely to be high and little explored. Broadly speaking, the processes that we will focus on include capsid disassembly (uncoating), reverse transcription, evasion of innate immune factors, and nuclear entry. Given the importance of the capsid structure and its interactions for many of these processes, we have expanded capsid studies to include an analysis of CA protein maturation and capsid formation. We plan to build on our successes and apply the extensive and complementary experimental expertise of our team to carry out 1) biochemical and high-resolution structure studies of individual proteins and complexes, 2) proteomics analyses to identify novel interactions and complexes, 3) virology and imaging studies to understand protein function in the context of the cell and virus infection, 4) sequence analyses of evolutionary correlations in protein interactions and dynamics, and 5) computational analyses to generate an all-atom model of the HIV-1 capsid and to elucidate the physical basis of uncoating (the process by which the capsid disassembles). Further, we plan to extend our Correlative Imaging Technology Development Program to develop specific probes and tools for exploring the dynamics of HIV-1 and associated proteins during cellular infection PUBLIC HEALTH RELEVANCE: Results provided by the proposed research are expected to have major implications in the global fight against AIDS, still considered an incurable disease with a pressing need for new therapeutic strategies and novel drug targets. Identifying and characterizing atomic structures of key HIV-1 host protein interactions in the immediate post-entry stage of the virus lifecycle will open new avenues in this endeavor.